Contrasting Genomic Variability between Clones from Field Isolates and Laboratory Populations of Schistosoma mansoni

The extent of genomic variability of clones of Schistosoma mansoni obtained from field isolates was compared with that of strains that have been laboratory maintained. Analysis was undertaken using randomly amplified polymorphic dNAs (RAPDs) generated with three primers. Phenograms showing the similarity among the clones were constructed. The data showed that while the laboratory strain is highly homogeneous the clones derived from the field populations were highly variable with 43% of RAPDs exhibiting polymorphisms among 23 clones. Clones isolated from the same infected individual were always more closely grouped than clones from different individuals. The data clearly demonstrated that earlier analyses of the genomic variability in S. mansoni have underestimated this phenomenon due to the failure to examine field isolates

Identification of Transcribed Sequences (ESTs) in the Trypanosoma cruzi Genome Project

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.

Genetic Data Showing Evolutionary Links between Leishmania and Endotrypanum

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Selection of Beauveria bassiana and Metarhizium anisopliae Isolates to Control Triatoma infestans

Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%), conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50) varied from 7.1x105 to 4.3x106 conidia/ml, for a B. bassiana isolate (CG 14) and a M. anisopliae isolate (CG 491) respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30°C, compared to 15 and 20°C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity).

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mnl I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails

Inter-specific and developmental differences on the array of antennal chemoreceptors in four species of triatominae (Hemiptera: Reduviidae)

The aim of the work was to investigate the pattern of chemoreceptor sensilla in adults and fifth stage nymphs of Rhodnius prolixus, R. neglectus, Triatoma infestans and T. sordida in order to study differences and similarities between genera and species. Three types of sensilla were analyzed by light microscopy: thin-walled trichoidea, thick-walled trichoidea and basiconica. The number of sensilla of each three types were counted. The length of the antennal segments were also used as a variable for the analysis. The statistical analysis showed that the number of these antennal chemoreceptors had significant differences between species and between adults and nymphs of each species. Discriminant analysis separates incompletely the fifth stage nymphs of the four species and showed similarity between them. Discriminant analysis performed with 12 variables of the antennae, allowed a complete separation of the adults of the four species.

New species records for the blackfly (Diptera-Simuliidae) fauna of Argentina with description of adults, pupa and larva of Simulium oyapockense s. l. and S. seriatum

Two blackfly species Simulium (Cerqueirellum) oyapockense Floch & Abonnenc and S. (Hemicnetha) seriatum Knab are recorded from Argentina, representing the most southern register for both species. S. oyapockense is a species epidemiologically very important, as a vector of onchocerciasis in the Amazonian focus. Both species are described and illustrated and their distribution are reported, in similarity to others like S. roraimense Nunes de Mello and S. ganalesense Vargas et al. in reference to S. oyapockense and S. mexicanum Bellardi similar to S. seriatum are discussed.

Borrelia-like spirochetes recovered from ticks and small mammals collected in the Atlantic Forest Reserve, Cotia county, State of São Paulo, Brazil

Forty-four marsupials, 77 rodents and 161 ticks were captured in an Atlantic Forest Reserve in Cotia county, State of São Paulo, where human cases of Lyme disease (LD) simile were reported. Twenty-one borrelia-like spirochete isolates were recovered from the mammals' blood and rodent livers or spleens, and triturated ticks inoculated into BSK II medium. Our results suggest that the reservoirs and ticks collected may harbor borrelia-like spirochetes, some of which have an antigenic similarity with the unknown causative agent of LD simile in Brazil, and/or with North American Borrelia burgdorferi s.s.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails.

Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found.

Recent studies on Schistosoma intercalatum: taxonomic status, puzzling distribution and transmission foci revisited

Schistosoma intercalatum, which causes human rectal schistosomiasis in Africa, still presents a great interest for its imprecise taxonomic status and its puzzling distribution in Africa. Two geographically isolated strains of S. intercalatum are recognized, the Lower Guinea strain and the Congo strain, which differ from each other in a number of morphological, biological and biochemical characteristics. Recent molecular data using RAPD markers indicate high divergence between the two strains, with values of Nei and Li's similarity indice allowing recognition of two genetically distinct taxa: experiments on pre- and post-isolating mechanisms are in progress in order to re-evaluate the taxonomic status of this polytypic species. With regard to its geographical distribution, S. intercalatum is characterized by the existence of two stable endemic areas (localized in Lower Guinea and North East of Democratic Republic of Congo) which correspond to the historical areas of species discovery, and the emergence during the last 15 years of new foci of the Lower Guinea strain outside previously known endemic areas. The absence of local adaptation of the Lower Guinea strain to its intermediate host, supported by experimental studies, may help to facilitate the spread of this strain. Nevertheless, the present restricted distribution of this species remains puzzling, because its potential snail hosts (bulinids) are widely distributed throughout much of Africa. Recent experimental and epidemiological studies suggest that interspecific sexual interactions between human schistosomes could have a role in limiting the distribution of S. intercalatum: the competitive sexual processes acting among human schistosomes show that S. haematobium and S. mansoni are always competitively dominant over S. intercalatum. These epidemiological observations lead the authors to distinguish three kinds of transmission foci for S. intercalatum.

Clinical and epidemiological aspects of human parvovirus B19 infection in an urban area in Brazil (Niterói city area, State of Rio de Janeiro, Brazil)

This study was designed to analyse the clinical and epidemiological data from human parvovirus B19 cases in a six-year study of rash diseases conduct in an urban area in Brazil (Niterói city area, State of Rio de Janeiro). A total of 673 patients with acute rash diseases were seen at two primary health care units and at a general hospital. A clotted blood sample was collected from all subjects at the time of consultation. Forty-nine per cent (330 cases) of the patients were negative for dengue, rubella and measles IgM or for low avidity IgG to HHV-6. Of these 330, 105 (31.8%) were identified as IgM positive to parvovirus B19 by using an antibody capture EIA. During the study period, three distinct peaks of parvovirus infection were detected, suggesting that the disease appears to cycle in approximately 4-5 years. B19 infection was characterized by variable combinations of fever, flu-like symptoms, arthropathy, and gastrointestinal symptoms. Frequency of fever and arthropathy was substantially higher in adults, 75% [chi2 (1 D.F.) = 11.39, p = 0.0007] and 62.5% [chi2 (1 D.F.) = 29.89, p = 0.0000], respectively. "Slapped-cheek" appearance and reticular or lace-like rash were seen in only 30.1% of the children. No adult presented this typical rash. The lack of the typical rash pattern in a large proportion of parvovirus B19 and the similarity of clinical manifestations to other rash diseases, specially to rubella, highlight the difficulty of diagnosing B19 infection on clinical grounds alone.